Determining the Harvest Time of Camu-Camu [Myrciaria Dubia (H.B.K.)

Scientia Horticulturae 186 (2015) 15–23

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Scientia Horticulturae

journal homepage: www.elsevier.com/locate/scihorti

Determining the harvest time of camu-camu [Myrciaria dubia (H.B.K.)

McVaugh] using measured pre-harvest attributes

a,∗ b,1 c

Leandro Camargo Neves , Vanuza Xavier da Silva , Edvan Alves Chagas ,

d e

Christinny Giselly Barcelar Lima , Sergio Ruffo Roberto

Federal University of Roraima (UFRR), Cauamé Campus, BR-174 Km 12, Monte Cristo, Boa Vista-RR, CEP: 69301-970, Brazil

Agronomy Graduate Program of the Federal University of Roraima (UFRR), Cauamé Campus, BR-174 Km 12, Monte Cristo, Boa Vista-RR,

CEP: 69301-970, Brazil

Embrapa Roraima, BR 174, km 08, C.P. 133, Industrial district, CEP: 69301-970, Boa Vista-RR, Brazil

Universidade Estadual de Londrina, UEL-CCA, Agronomy Departament, Campus Universitário, Jd. Perobal, PR - Brasil - Mailbox: 6001.,CEP: 86051990,

Londrina, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The characterization of the physical, chemical, and physiological parameters of camu-camu fruit at vari-

Received 30 October 2014

ous stages of development may help to determine the optimal harvest time of this fruit. Camu-camu fruits

Received in revised form 28 January 2015

from a rural property in the municipality of Cantá/RR were marked and harvested at 53, 60, 67, 74, 81, 88,

Accepted 5 February 2015

95, and 102 days after anthesis (DAA) during six seasons (years) of study. The following attributes were

measured: fresh weight, diameter, total soluble solids (TSS), starch, titratable acidity (TA), the TSS/TA

Keywords:

ratio, total and reducing sugars, total and soluble pectins, pectic enzymes, vitamin C content, and the

Maturity stages

production of CO2 and ethylene. The combination of the absence of a climacteric peak in the respiratory

Qualitative attributes

pattern and the low and invariable production of ethylene during development indicates that camu-camu

Ascorbic acid

fruit is non-climacteric. The best harvest time for this fruit occurred between 88 and 95 days. During this

Amazon fruit

period, measured parameters such as coloration (85% and 100% red, respectively), high TSS:TA ratio,

reduced TA, and high TSS, reducing sugar, soluble pectin, and vitamin C concentrations achieved their

optimal.

1. Introduction As mentioned, camu-camu fruits have the highest vitamin C

content of all fruits studied to date (Alves et al., 2002; Rodrigues

Camu-camu is an Amazonian fruit that deserves attention, in et al., 2001; Yuyama et al., 2002). Yuyama et al. (2002) measured

general, for its content of vitamin C. Commercially, the recent intro- very high concentrations of vitamin C in fruits from a natural habi-

duction of this fruit into the world market, especially in America tat in the eastern portion of Roraima, Brazil, ranging from 3571 mg

and in centers of high consumption of fruits such as Europe and of ascorbic acid/100 g fresh pulp in the Rio Mau region to 6112 mg of

Asia, has aroused great interest by local farmers. In this sense, ascorbic acid/100 g fresh pulp in the Rio Tacutu region. These sur-

camu-camu has awakened interests of industrial sectors such as pass those measured in the acerola (1790 mg/100 g pulp), a fruit

the pharmaceutical, cosmetics, and food industries due the natural that was previously considered to have the highest of vitamin C.

bio compounds that may be the extracted from its skin and pulp Because the camu-camu is very high in vitamin C, a vitamin that

and, possibly, with high antioxidant activities (Smiderle and Sousa, is not synthesized by the human body and must therefore be con-

2008). sumed in the diet, there is a strong demand for this fruit in the

natural products market.

The vitamin C content, similar to other phytochemical proﬁles

of the camu-camu, is known to change throughout the fruit’s stages

∗

Corresponding author. Tel.: +55 95 3627 29 03; fax: +55 95 3627 29 03. of maturation. The occurrence of the ripening stage, in particular,

E-mail addresses: [email protected] (L.C. Neves), [email protected]

signiﬁcantly affects the physical characteristics and chemical

(V.X.d. Silva), [email protected] (E.A. Chagas), [email protected]

composition of the camu-camu (Andrade et al., 1995). Camu-camu

(S.R. Roberto).

1 fruits are green when unripe and turn red or purple during

Tel.: +55 95 3627 29 03; fax: +55 95 3627 29 03.

http://dx.doi.org/10.1016/j.scienta.2015.02.006

16 L.C. Neves et al. / Scientia Horticulturae 186 (2015) 15–23

maturation and ripening because of the presence of anthocyanins divided in two groups:(a) Destructive analysis: three replicates of

(Zanatta et al., 2005). According to Yuyama et al. (2011), red 10 fruits weighing 92.0 ± 36.0 g; and, (b) Non-destructive analysis:

camu-camu fruits have greater vitamin C, but other studies have likewise, approximately 90 fruits (weighing 828.8 ± 319.0 g were

reported higher concentrations in unripe fruits (Alves et al., 2002). divided into three replicates of 30 fruits for the measurement of

This discrepancy is the main obstacle to establishing the commer- CO2 and ethylene production, and these fruits were kept intact until

cial production of camu-camu in Brazil because numerous small the end of the experiment.

producers use skin color as the only indicator for determining the Peels and pulps, without seeds, from each replicate, were mixed

optimal time of harvest. Notably, the timing of the harvest can be and crushed to obtain a commingled mass of the whole fruit. Only

critical for maintaining high concentrations of vitamin C and other for the vitamin C analysis that peel and pulp were set apart and

biomolecules. measured separately.

The ideal ripening stage for harvesting camu-camu can be All the evaluations were performed weekly and, measured no

determined by evaluating maturation and ripening curves that more than 50 ± 5 min after each speciﬁcally harvest time. Physical

consider chemical and physical changes, such as weight loss and measurements, total soluble solids content, and titratable acidity,

changes in the total soluble solids, titratable acidity, and vitamin as well as the collect and analysis of gases (CO2 and ethylene),

C (Giovannoni, 2004). To produce these curves, physical, chemical, were performed immediately after each harvest. For the other

and physiological parameters of the fruit are measured throughout analysis, were prepared samples (25–50 g of each replicate) that

◦

their development (Andrade et al., 1995). were frozen at −40 C. The frozen samples were used to measure

The aim of the present study was to characterize the physi- the starch, total and reducing sugars, total soluble pectins, and

cal, chemical, physico-chemical, and physiological parameters of pectic enzymes for each period. About 20 g of each replicate were

◦

camu-camu fruits during various stages of development to deter- lyophilized at 200 ␮m Hg and −40 C and stored in a freezer for

mine the ideal harvest time of camu-camu in the state of Roraima. purposes of preservation. Analyses of vitamin C concentrations

were performed in the Plant Bioactive and Bioprocessing Research

Laboratory TAMU/USA.

2. Materials and methods

The following analyses were performed in all treatments (fresh

fruits):

The fruits used in this experiment were harvested during six

seasons (years) of study from a rural property located in the

county/state of Cantá/Roraima (RR), near one of the Branco river, 2.1. Fresh pulp (g)

60 km from Boa Vista (the state capital).

To obtain fruits from the entire camu-camu population, approxi- The mass of fresh pulp was measured by weighing 10 fruits (ran-

mately 2000 inﬂorescences from 40 plants (representing 90% of the domly chosen) from each replicate and dividing the total weight by

plants in the area) were marked. However, because a single inﬂo- the number of fruits (10), thereby obtaining the mean of individual

rescence can contain materials at various stages, buds at later stages fresh weight.

(developing ovaries) and earlier stages (young buds that were only

a few days old) were removed when necessary, thereby leaving only 2.2. Polar and equatorial diameter (mm)

the buds at the previously described stage of interest. These ﬂoral

buds were marked with colored wire and a label containing the date A digital caliper was used to measure the polar and equatorial

and an ID number was attached. The ﬂoral buds were monitored diameters of 10 fruits per replicate. The equatorial diameter was

weekly from the date of marking until the start of fruit harvesting. measured horizontally, and the polar diameter was measured ver-

Fruits were harvested every seven days, starting from the anthe- tically from pole to pole using the location of the stem cavity as a

sis of the inﬂorescences (Fig. 1). Fruits were harvested at eight time reference point.

points (53, 60, 67, 74, 81, 88, 95, and 102 days after anthesis–DAA).

Approximately 120 fruits, representative of the entire popula- 2.3. CO2 and ethylene production

tion, were collected at each harvest. On the day of the harvest,

the fruits were placed in plastic pots inside Styrofoam boxes CO2 and ethylene production analyses were performed accord-

containing ice and then transported to the Laboratory of Food ing to the methods described by Prill et al. (2012). Brieﬂy, 10

Technology–UFRR and; care was taken to avoid direct contact fruits weighing 92 ± 35.54 g (approximately 0.1 kg per replicate)

◦

between the fruit and the ice. In the laboratory, the fruits were were placed in hermetic containers for 1 h at 22 ± 1 C; 5 mL of the

◦

cleaned using pure water and dried at room temperature (25 2 C gaseous atmosphere was then collected from each container (treat-

and 75 2% R.H.), allowing the excess water to evaporate natu- ment) with a hypodermic syringe to measure the production of

± rally ( 2 h). After the fruits were cleaned, 30 samples (weighing ethylene and CO2. Ethylene concentrations were quantiﬁed by gas

276.3 106.0 g) were randomly selected for physical analysis and chromatography using a Varian gas chromatograph, model 3300,

Fig. 1. Visual appearance of camu-camu fruits [Myrciaria dubia (H.B.K.) McVaugh] at various stages of development. The numbers inside the pictures correspond to the

numbers of days after anthesis (DAA).

L.C. Neves et al. / Scientia Horticulturae 186 (2015) 15–23 17

equipped with a 1/8” stainless steel column packed with Porapak 2.10. Pectin methyl esterase (PME) and polygalacturonase (PG)

N and a ﬂame ionization detector. The CO2 concentrations were

measured on a Shimadzu CR 950 chromatograph equipped with Pectin methyl esterase (PME) activity was measured according

a thermal conductivity detection system. Ethylene (100 ppm) and to the method described by Jen and Robinson (1984). Brieﬂy, we

CO2 (5%) gases were used as standards. The results were expressed measured the ability of the enzyme to catalyze the demethylation of

␮

in mg of CO2/kg h and in ␮L of ethylene/kg h. pectin, i.e., to produce 1 mol of NaOH per minute under the assay

conditions. The results are expressed in ␮mol of NaOH/g min. Poly-

galacturonase (PG) activity was measured according to the method

2.4. Total soluble solids (TSS)

described by Pressey and Avants (1973). Brieﬂy, we measured the

ability of the enzyme to catalyze the formation of 1 ␮mol of reduc-

The level of TSS was measured by refractometry on a portable

ing sugars per minute per gram of fruit pulp. The results were

refractometer, with a temperature correction. A drop of pure juice

expressed in U.A.E/g min.

from each of the three replicates containing 30 fruits was measured.

◦

The results were expressed in Brix (IAL, 2008).

2.11. Vitamin C

High-performance liquid chromatography (HPLC) analysis was

2.5. Ratio of total soluble solids (TSS) to titratable acidity (TA)

performed to measure the vitamin C (ascorbic acid) level according

to the method described by Campos et al. (2009) using a Shimadzu

The SS/TA ratio was calculated as the ratio of the level of total

LC VP HPLC system with a pump (LC-6AD), a UV-VIS detector (SPD-

soluble solids to that of the titratable acidity.

10AV VP), and a YMC-Pack ODS column (250 × 4.6 mm, 5 mm i.d.).

The separation of vitamin C was accomplished by isocratic elution

2.6. Titratable acidity (TA) using a phosphoric acid solution at pH 3 as the mobile phase. The UV

detector was set at 254 nm. The quantiﬁcation of the compounds of

The titratable acidity was measured by diluting 10 mL of pure interest was based on the peak area. The results were expressed in

juice in 100 mL of distilled water and titrating this solution with mg/100 mL of pulp and peel. A 10 g sample was extracted in 10 mL

0.1 M NaOH to pH 8.1. The results were expressed in g of citric of water adjusted to pH 1.5 with 10 mL of phosphoric acid in water

acid/100 g of pulp (IAL, 2008). (2% v/v). The extracts were ﬁltered through ﬁlter paper, and 1.5 mL

of buffer (0.01 MKH2PO4, pH 8.0) was added to 1.5 mL of the ﬁltered

sample extract. Subsequently, 1.0 mL of this mixture was loaded

2.7. Total and reducing sugars

into a C18 cartridge; 3 mL of water (pH 1.5) adjusted with 2 mL

of aqueous phosphoric acid (2% v/v) was passed through the car-

The total and reducing sugars were measured according to the

tridges and 20 mL of eluent was injected into the HPLC. The results

method of Nelson (1944), and the results were expressed in mg

were expressed in mg of ascorbic acid/100 g of sample on a dry basis

of glucose/100 g of pulp. A calibration curve using glucose as the (d.b.).

standard was created to calculate the total and reducing sugars.

2.12. Statistical analysis

2.8. Starch

A preliminary exploratory analysis of the data was conducted.

The data were normally distributed and the errors were indepen-

One gram of previously lyophilized sample was weighed into

dent and exhibited homoscedasticity. The design was completely

a 250 mL Erlenmeyer ﬂask, and 50 mL of 1 M HCl (8.5 mL of HCl

randomized with a factorial scheme only for vitamin C analysis

in 1 L of distilled water) was then added to the ﬂask. The ﬂasks

(seven harvest times × two sample types (pulp and peel,). Three

were sealed with cotton plugs wrapped in self-adhesive plastic ﬁlm.

replicates of 10 fruits analyzed used for the physical analyses, while

The ﬂasks containing the samples were placed in microwave-safe

three replicates of 30 fruits were analyzed for the chemical anal-

plastic containers that contained sufﬁcient water to prevent the

yses. Data were subjected to an analyses of variance (ANOVA),

desiccation of the samples. With the ﬂasks inside, the microwave

and the means were compared by Tukey’s multiple range test and

was operated for 20 min at full power. After this period, during

least signiﬁcant differences (LSD) at P ≤ 0.05. All statistical anal-

which the starch was converted to sugar, a few drops were removed

yses were performed using the SISVAR – UFLA program, version

for testing with Lugol (iodine in potassium iodide, which turns the

5.1.

solution yellow). The sample was then neutralized with 10% NaOH

(100 g NaOH/L of water), and three drops of phenolphthalein were

3. Results and discussion

added as an indicator (to ensure that the solution remained light

pink). Because the sugar in the analyzed sample was not previ-

All data presented here represent the means of six seasons

ously separated from the sample, the percentage that corresponds

(years) of study.

to sugar was subtracted from the measurement to calculate the

ﬁnal starch content (IAL, 2008). The results were expressed in mg

3.1. Fresh weight and polar and equatorial diameters of fruits

of glucose/100 g of pulp.

In a natural habitat, the camu-camu develops over a 102-day

2.9. Total and soluble pectins period of full ﬂowering and ripening. The fresh weights of the camu-

camu fruits increased between 53 and 81 days after anthesis (DAA)

These compounds were extracted following the procedure and declined thereafter; the weight of these fruits peaked at 12 g

described by McCready and McCoomb (1952) and measured col- (Fig. 2).

orimetrically by performing acarbazole reaction following the Thus, the developmental period of the fruit was characterized

technique described by Bitter and Muir (1962). The total and sol- by an initial period of rapid growth that lasted until 60 DAA; during

uble pectins were expressed as the percentage (%) of galacturonic this time, the weight of the fruits tripled from an initial weight of

acid/100 g of pulp. 2.11 g to a mean fresh weight of 6.89 g. This increase corresponds

18 L.C. Neves et al. / Scientia Horticulturae 186 (2015) 15–23

15.00 30.00 ab a ab a bc d b cd 12.00 b 25.00 c e ab a ab c bc ight 20.00 f c 9.00 d ) c we d g) d

( 15.00 mm

6.00 ( e 10.00 Fresh 3.00 e 5.00

0.00 0.00

Equatorial and polar diameter

53 60 67 74 81 88 95 102 53 60 67 74 81 88 95 102

Days after anthesis (DAA) Days after anthesis (DAA)

Entire fruits Polar diameter Equatorial diameter

Fig. 2. Fresh weights (g) of camu-camu (Myrciaria dubia) fruit during development Fig. 3. Equatorial and polar diameters (mm) of camu-camu (Myrciaria dubia) fruit

from 53 to 102 DAA. during development from 53 to 102 DAA.

The patterns of growth for the polar and the equatorial diam-

to a growth rate of 683.00 mg/day. The fruits were still green (i.e.,

eters were similar (Fig. 3); however, the growth of the equatorial

immature) during these initial two evaluation periods (Table 1).

diameter exceeded that of the polar diameter during the devel-

From 67 to 81 DAA, the fruits gained weight at a slower rate, and

opment and ripening of the fruits. This ﬁnding suggests that the

the peel began to turn red; at the end of this period (81 DAA), the

width of the fruit is greater than the length, which is a deﬁning

fresh weight reach the maximum, and 50% of the peel area of these

characteristic of the sub globose shape of the camu-camu. Alves

fruits had acquired a reddish color (Table 1 and Fig. 1). These results

et al. (2002) reported that fruits harvested from the experimental

suggest that the fruit reached physiological maturity 81 DAA and

station of Embrapa Amazônia East had a similar sub globose shape,

ripened during the subsequent period.

with a mean equatorial diameter ranging from 22.31 to 23.06 mm

Fruit growth ceased by 81 DAA, and the mean weight of the fruits

and a mean polar diameter ranging from 21.44 to 22.11 mm in

declined until 88 DAA because of fruit transpiration (loss of turgor)

the fully green and fully red stages, respectively. The equato-

associated with the high insolation and evaporation characteristics

rial diameters of the camu-camu fruits analyzed in the present

of the Amazon region and the onset of ripening and senescence. A

study were greater than the equatorial diameters of camu-camu

similar pattern of growth has been reported for ‘Haden’ Mangos:

fruits from Mirandópolis (21.7–24 mm) and Iguape (São Paulo)

these fruits attained their maximum fresh weight 55 days after

(22.31–23.06 mm) (Zanatta et al., 2005). These ﬁndings suggest that

ﬂowering (DAF) (in a cycle lasting from 15 to 73 DAF) and exhib-

the soil and climatic characteristics of the Amazon are favorable for

ited fresh weight decreases and water losses thereafter (Castro Neto

the rapid growth and high yield of camu-camu fruit.

and Reinhardt, 2003). ‘Obatã’ coffee fruits also exhibited losses in

fresh weight and volume after the growth phase (Cunha and Volpe,

2011). 3.2. Respiration and ethylene production

The color of the fruits began to change from red (85% red) at

88 DAA to purple (100% purple 102 DAA). This change supposedly The mean respiratory rate of the camu-camu ranged from 10.00

reﬂects ripening and the onset of senescence. Yuyama et al. (2002) to 20.00 mg CO2/kg h, which corresponds to an intermediate respi-

has described ripened fruits as being purplish or almost black, ration rate (Kader, 2002). The respiration rate of camu-camu fruits

slightly dehydrated, and shriveled. decreased throughout fruit development (Fig. 4).

Following a similar pattern as the fruit fresh weight, the equa- The largest amounts of CO2 were produced during the ﬁrst

torial and polar diameters of the camu-camu fruit increased until evaluation periods (53, 60, and 67 DAA) because of the high

81 DAA and then decreased (Fig. 3) because of a loss of turgor. metabolism associated with initial fruit development during these

The growth in the diameters of the fruits was proportional to periods (Payasi and Sanwal, 2010). Steady decreases in CO2 pro-

the gain in fresh weight and was characterized by higher growth duction were observed during the subsequent periods, and there

rates during the initial evaluation period and a deceleration and was no detectable respiration after 81 DAA, when the fruits had

stabilization of the growth by 81 DAA. reached physiological maturity. In a study of camu-camu fruits in

Table 1

Color of the peel of camu-camu fruit during development. 25.00 5.00

a Eth

22.50 4.50 (µL

Days of anthesis (DAA) Peel color y / h) 20.00 b 4.00 b of lene kg

53 DAA 100% green eth

/ 17.50 3.50

60 DAA 100% green y 15.00 3.00 lene production CO c

67 DAA 90% green with slight reddish 12.50 c c 2.50 production /

coloration on the side exposed 2 10.00 2.00

to the sun mg CO a abc ab 7.50 bc bc c d 1.50 / (

d h)

74 DAA 75% green with slight reddish 5.00 1.00

coloration on the side exposed

2.50 d d 0.50

to the sun

0.00 0.00

81 DAA 50% green and 50% red on the

53 60 67 74 81 88 95 102

side exposed to the sun

88 DAA 85% red with some light green Days after anthesis (DAA)

on the side not exposed to the CO2 Ethylene

sun

95 DAA 100% red

Fig. 4. Production of CO2 and ethylene in camu-camu (Myrciaria dubia) fruit during

102 DAA 100% dark red to purple

development from 53 to 102 DAA.

L.C. Neves et al. / Scientia Horticulturae 186 (2015) 15–23 19

Fig. 5. Titratable acidity (5.1) and soluble solids content (5.2) of camu-camu fruit during development from 53 to 102 DAA.

the Colombian Amazon conducted from anthesis to full maturity Amazon, Pinto et al. (2013) detected a climacteric behavior for

by Bardales et al. (2008), a similar pattern was observed, also char- the camu-camu fruits grown in an upland area. Nevertheless, in

acterized by no respiratory peak and the maintenance of moderate the present study, the fruits were harvested in their native region,

respiratory rates until the end of the experiment, contradicting the characterized by seasonal ﬂooding, based on temporal criteria. In

results presented by Pinto et al. (2013). The Camu-camu fruit is addition, using a subjective parameter (such as the skin color)

considered to be a non-climacteric fruit because of the lack of a to determine the harvest time may cause the producer to make

respiratory peak before and/or during ripening, (Payasi and Sanwal, an incorrect decision regarding the optimal harvest time because

2010). In this regard, by 95 DAA, CO2 production decreased to below of the inherent color variation in the fruit (especially for a non-

5.00 mg CO2/kg h, possibly because of the late harvest time and the domesticated species), even at the (supposedly) same maturity

senescence process of the fruits. stage. Wills et al. (1998) also reported that the respiration rate

The camu-camu fruit is also considered non-climacteric because decreases in non-climacteric fruits during ripening and that the

of its low and invariable production of ethylene during develop- biochemical transformations responsible for ripening occur more

ment and ripening (Fig. 4). Our data support the ﬁndings of Bardales slowly. Therefore, unlike climacteric fruits, which have the ability

et al. (2008), who compared the respiratory rates of various non- to ripen after harvest, non-climacteric fruits (such as the camu-

climacteric species of the Myrtaceae family and determined that camu) only ripen when attached to the plant (Payasi and Sanwal,

camu-camu was characterized by a moderate respiratory rate, no 2010).

detectable climacteric peak, and considerable ethylene production

during development. Other authors (Pinedo et al., 2011; Andrade 3.3. Titratable acidity (TA), total soluble solids (TSS), and TSS/TA

et al., 2010) who have conducted studies of camu-camu fruits culti- ratio

vated in the Amazon region have also presented results that suggest

non-climacteric behavior for the camu-camu fruit. The titratable acidity of the camu-camu fruits decreased until 88

According to Pinto et al. (2013), camu-camu fruits were con- DAA to a minimum of 2.28 g of citric acid/100 g and then increased

sidered to be climacteric because fruits that were harvested while slightly but non-signiﬁcantly at 95 and 102 DAA (Fig. 5).

unripe (stage 1) could exhibit climacteric and ethylene production The decrease in TA can be explained by the use of organic acids

peaks after harvest. However, despite these characteristics, these in respiration and as a result of a dilution effect occurring during

fruits did not develop several of the quality attributes expected fruit growth (Prassana et al., 2007). The small increase in TA during

for climacteric fruits, and these attributes were only observed in the ﬁnal two collection periods (95 and 102 DAA) was associated

fruits harvested at more advanced maturation stages. Thus, based with the loss of moisture in the fruit and the consequent concen-

on these conditions, the results obtained by these authors can be tration of the acids in the cellular ﬂuid. This pattern is consistent

considered as contradictory because unripe fruits that are classiﬁed with previous ﬁndings that completely green fruits have high TA,

as climacteric should develop all quality attributes, also exhibited and completely red fruits have relatively low TA (Alves et al., 2002;

by ripe fruits. So, either the fruits were harvested too early, or Pinto et al., 2013).

they exhibited a non-climacteric pattern, given that post-ﬂowering The TSS content of the camu-camu fruits decreased to a mini-

◦

or post-anthesis monitoring was not performed to determine the mum of 5.00 Brix at 74 DAA and then increased from 81 to 102 DAA

◦

developmental stage at which the fruits were harvested. According to a ﬁnal value of 6.71 Brix (Fig. 5.2). The decrease in the ﬁrst stage

to Fonseca et al. (2002), the maturation stage usually inﬂuences the of fruit development may have occurred because sugars were used

level of respiratory activity. Thus, very unripe or non-climacteric to form structural polysaccharides (Rossetto et al., 2004) and com-

fruits exhibit accelerated metabolism and high respiratory activity. ponents of cell walls. The increase in TSS content that began 81 DAA

When removed from the parent plant, these fruits tend towards resulted from increases in the reducing sugars associated with the

senescence rather than maturation. onset of maturation. Camu-camu fruits at green and reddish-green

In turn, Bardales et al. (2008) monitored the development of stages (unripe) also contained lower total soluble solids compared

camu-camu fruits from the full ﬂowering stage until fruit ripening. to those at red-greenish or purple stages, which are considered fully

These authors found that the fruits exhibited high respiration rates ripe fruits in the study by Pinto et al. (2013).

during the initial stages of development, with this rate decreasing However, in the case of the camu-camu, organic acids also con-

after the fruits reached their maximum growth and remaining at tribute to the increase in TSS (Alves et al., 2002). Thus, the increase

a moderate level of approximately 100 mg of CO2/kg h or less dur- in TA during the ﬁnal two periods may have also contributed to the

ing ripening. Furthermore, in that study, the pattern of ethylene increase in TSS. Alves et al. (2002) detected no signiﬁcant differ-

production during ripening was compatible with a non-climacteric ences in the TSS contents of completely green and completely red

pattern, a result that conﬁrms that the respiratory pattern of camu- fruits. We also failed to detect signiﬁcant differences between the

camu is similar to that of other non-climacteric species of the family TSS contents of camu-camu fruits at 53 DAA and 102 DAA (Fig. 5.2).

Myrtaceae. The TSS contents of the fruits increased when the fruits began

However, in camu-camu fruits cultivated in the São Paulo region, to acquire a completely red peel (i.e., at the onset of fruit ripening).

where the soil and climatic characteristics differ from those of the Thus, 81 DAA appears to mark the end of physiological maturity

20 L.C. Neves et al. / Scientia Horticulturae 186 (2015) 15–23

3.00 a of this fruit for the processing for derivatives such as jams, juices,

bc b b and ice cream, which can add value to this fruit. 2.50 cd

2.00 d d 3.4. Total sugars, reducing sugars and starch

1.50

The total and reducing sugars increased during the development

of the camu-camu fruit, with a more pronounced accumulation of

SS/ 1.00

reducing sugars (Fig. 7.1).

0.50 The increase in the total and reducing sugars coincided with

the increases in the fresh weight and diameter of the fruits; this

0.00

period represents the period when the fruits are nearly ripe and,

53 60 67 74 81 88 95 102 consequently, the ideal harvest time. The concentration of total

Days after anthesis (DAA) sugars peaked at 1.68 mg glucose/100 g of sample at 88 DAA and

then declined during the ripening process as these sugars were

SS/TA

metabolized. An increase in total sugar during the transition from

the green stage to the fully red stage was also reported by Alves

Fig. 6. Ratio of total soluble solids to titratable acidity (TSS/TA) in camu-camu fruit

et al. (2002). The breakdown of sugars also resulted in an increase

(Myrciaria dubia) during development from 53 to 102 DAA.

in the reducing sugars to a maximum of 0.95 mg glucose/100 g at

102 DAA. This period was considered to represent the period when

the fruit was most mature and when fruit senescence began. The

and the onset of ripening, and the TSS contents of the fruit differed

production of total and reducing sugars peaked in jabuticaba fruit

signiﬁcantly in the two harvest periods following this transition

at 55 DAA (i.e., during the period of fruit ripening) and decreased in

period. It can thus be inferred that TSS can be used to measure

subsequent periods when these sugars were used for respiratory

the degree of maturity of ripening camu-camu fruits. For camu-

processes (Corrêa et al., 2007). Such decreases in sugar were not

camu fruits, an association between the SS and the color of the peel

observed in the camu-camu because the respiratory rate decreased

should not be ruled out; indeed, when the fruit was completely

during the ﬁnal periods of fruit ripening.

purple (102 DAA), the TSS content of the fruit was greatest. The

The sugar patterns suggest that the synthesis of sugars (espe-

pulp of the camu-camu fruit became sweeter and less acidic as

cially reducing sugars) was low during the initial period of fruit

ripening advanced. Therefore, the ripening period represents an

development until 67 DAA. The increased sugars in subsequent har-

ideal harvest time for the in natura consumption of this fruit or the

vests coincided with steadily declining starch contents (Fig. 7.2).

processing of fruit for derivatives such as jams and jellies.

Alves et al. (2002) also reported a decrease in the starch content

Because the TSS contents and the TA were inversely related dur-

of mature camu-camu fruits. In contrast, the starch content, but

ing maturation, the TSS/TA ratio exhibited a positive linear increase

not the sugar, of jabuticaba decreased from 25 DAA until 50 DAA

(Fig. 6).

(Araújo et al., 2010). The reducing sugars fructose and glucose come

This ratio is an important qualitative attribute because it indi-

from the degradation of sucrose and polysaccharide reserves such

cates the relative contributions of the compounds responsible for

as starch (Prassana et al., 2007). Thus, a lower starch content and

sweetness and acidity, respectively, and therefore provides an indi-

higher level of reducing sugars is associated with a more advanced

cation of the ﬂavor of the fruit (Prassana et al., 2007).

stage of fruit ripening. Although the reducing sugars increased

In addition, this ratio is considered a marker of the maturation

during fruit development after 81 DAA, they remained high and

stage of fruit that can predict the sweetness of the fruit. Stenzel et

relatively stable until 102 DAA, in contrast to the starch contents.

al. (2006) also observed a positive linear progression in the TSS/TA

Therefore, the starch contents could also be used to estimate the

ratio of fruits of ‘Folha Murcha’ orange trees grafted on various vari-

degree of maturity of camu-camu fruits and to determine the ideal

eties of stocks from two distinct regions; these authors used this

harvest time of non-climacteric fruits such as the camu-camu.

ratio to estimate the ideal harvest time. The TSS/TA ratio can also

be used to assess the degree of maturation of the camu-camu. The

TSS/TA ratios observed in our study were relatively low, ranging 3.5. Total and soluble pectins and pectic enzymes

from 1.79 at 53 DAA to 2.75 at 102 DAA, indicating that the camu-

camu fruit is not as sweet as other fruits such as the ‘Paluma’ guava The degradation of cell wall polysaccharides, which is mea-

(TSS/TA ratios ranging from 9.88 to 17.66) (Cavalini et al., 2006) or sured by the total and soluble pectins, may have contributed to

the ‘Dixie’ grape (SS/TA ratios ranging from 0.51 to 6.88) (Sachi and the changes in the reducing sugars (Fig. 8.1).

Biasi, 2008). This ratio also conﬁrms that the camu-camu fruit has The concentration of total pectins tended to decrease in a linear

an acidic flavor, even during ripening. This acidity limits the con- manner; this finding confirms that the first experimental har-

sumption of this species in natura and favors the dilution and use vests (53, 60, and 67 DAA) coincided with the fruit’s stages of

Fig. 7. Total and reducing sugars (7.1) and starch (7.2) in camu-camu (Myrciaria dubia) fruit during development from 53 to 102 DAA.

L.C. Neves et al. / Scientia Horticulturae 186 (2015) 15–23 21

Fig. 8. Total and soluble pectins in camu-camu (Myrciaria dubia) fruit during development from 53 to 102 DAA (8.1) and; pectin methyl esterase (PME) and polygalacturonase

(PG) enzyme activity in camu-camu (Myrciaria dubia) fruit during development from 53 to 102 DAA (8.2).

physical growth during which the total pectin were high because A number of studies have reported increases in ascorbic acid

they contribute to the structure of young cells and tissues. Dur- during the maturation and ripening of the camu-camu fruit. Zapata

ing subsequent harvests, the depolymerization and consequent and Dufour (1993) reported an increase from 8.64 g of ascorbic

solubilization of pectins, together with other chemical changes, sol- acid/kg of fruit in unripe fruits to 9.70 g of ascorbic acid/kg of fruit in

ubilized the protopectin fraction, as evidenced by the steady decline completely red and fully ripe fruits. Andrade et al. (1995) measured

in total pectin and the slight increase in soluble pectin (Fig. 8.1). The the variation in vitamin C content during the development and six

increased of soluble pectins were a marker of the solubilization of stages of maturation of the camu-camu fruit and reported that vita-

pectins from the cell wall and the resultant structural loss of cellu- min C accumulated in the fruit from maturation stage 3 to the ﬁnal

lar tissues; Araújo et al. (2010) have noted similar ﬁndings. Alves stage (from 2004.66 to 2605.76 mg of ascorbic acid/100 g of fruit).

et al. (2002) have also reported decreases in the total pectins and a Alves et al. (2002) also measured higher ascorbic acid in red fruits

non-signiﬁcant increase in soluble pectin in the camu-camu during (up to 2061.04 mg of ascorbic acid/100 g of sample) than in green

the transition from the fully green to the fully red stage. According fruits (1910.31 mg of ascorbic acid/100 g of sample) and reported

to Araújo et al. (2010), the soluble pectin in the pulp and peel of that the mature camu-camu had higher vitamin C than other vita-

jabuticaba also tend to increase with maturation. min C-rich fruits, such as the acerola. Yuyama et al. (2011) also

Depolymerization occurred to the greatest extent during the determined that redder camu-camu fruits have higher vitamin C. In

period beginning 74 DAA; at this time, the activity of pectin methyl contrast, we found that younger fruits (88 DAA) had higher concen-

esterase (PME) also peaked and the activity of polygalacturonase trations of vitamin C than fully ripened fruits (102 DAA). Other stud-

(PG) began to increase. This period presumably corresponds to the ies have also reported decreases in ascorbic acid in more mature

onset of the cell wall degradation process and the consequent phys- camu-camu fruits. Chirinos et al. (2010) reported that completely

ical changes in the fruit (Fig. 8.2). This result conﬁrms that the onset green fruits had higher concentrations of ascorbic acid than com-

of ripening, which is marked by enhanced enzymatic activity in pletely red fruits. Smiderle and Sousa (2008) reported that unripe

the camu-camu, occurs when the fruit begins to take a reddish and ripe fruits had 2520.00 and 2590.00 mg/100 g of vitamin C in

coloration, as shown in Table 1. After this period, the activity of the samples, respectively, and concluded that fruits at these two

PME and PG steadily decreased and increased, respectively, until stages of maturity did not have signiﬁcantly difference in the vita-

102 DAA. A similar pattern occurred for the demethylation of poly- min C. Consistent with the results of our study, Correa et al. (2011)

mers of galacturonic acid, which is initially triggered by PME, and measured vitamin C in both the peel and pulp of ripening camu-

the subsequent cleavage of these polymers by PG (Prassana et al., camu fruits (also by HPLC) and found that vitamin C decreased as

2007). The activities of PME and PG in the present study were sim- the fruit ripened. Villanueva-Tiburcio et al. (2010) analyzed camu-

ilar to those reported by Alves et al. (2002), who determined that camu peels on a dry basis and found that fruits in an intermediate

PME activity was signiﬁcantly higher than PG activity in green fruits stage of ripening had higher ascorbic acid (53.49 ± 9.40 mg ascor-

and that PG activity were more pronounced in mature red fruits. bic acid/g d.b.) than fully ripe fruits (16.41 ± 3.64 mg ascorbic acid/g

These changes in the solubility of pectins during ripening are asso- d.b.) and completely green fruits (15.38 ± 5.81 mg ascorbic acid/g

ciated with the depolymerization of pectic chains and may lead to d.b.); however, the ascorbic acid in these fully ripe and green fruits

changes in the external appearance of the fruit. did not differ signiﬁcantly. In the present study, fruits at an inter-

mediate stage of maturation or that had just begun to ripen (88

DAA) also showed relatively high ascorbic acid that decreased in

3.6. Vitamin C

The pulp and the peel of camu-camu fruits were worked, pre- 6000

5500 a

sented high vitamin C (Fig. 9). 5000

The maturation phase was characterized by the accumulation 4500 b a b

4000 c c

of ascorbic acid and a higher rate of synthesis. During this phase, 3500

the volume of the fruits peaks, and the ripening and biochemical 3000 d d b b e

2500 d c

reactions that result in changes in the sensory compounds of fruits 2000

(which make the fruits edible) are initiated (Giovannoni, 2004). 1500 g f

1000

Synthesis activities peaked at 88 DAA (i.e., at the onset of ripen- (mg /100mL of sample)

Ascorbic acid content 500

ing) when the fruits had also accumulated the greatest amount 0

of ascorbic acid (4752.23 and 5178.49 mg of ascorbic acid/100 g of 53 60 67 74 81 88 95 102

sample in the pulp and peel, respectively). At the subsequent har- Days after anthesis (DAA)

vests, the ascorbic acid contents decreased in both fractions, most

Pulp Peel

likely because of the onset of catabolic reactions promoted by fruit

ripening. Similar results were also reported by Villanueva-Tiburcio

Fig. 9. Ascorbic acid in the samples of pulp and peel (lyophilized) of camu-camu

et al. (2010).). (Myrciaria dubia) fruit during development from 53 to 102 DAA.

22 L.C. Neves et al. / Scientia Horticulturae 186 (2015) 15–23

subsequent periods (Fig. 9). Villachica (1996) also reported that productive systems in the Colombian Amazonian region. Acta Hortic. 773,

173–178 (The Hague).

100% green, 100% ripe, and over-ripened fruit had 17%, 9%, and 20%

Bitter, T., Muir, H.M., 1962. A modiﬁed uronic acid carbazolereaction. Anal. Biochem.

less ascorbic acid, respectively, than fruits at an intermediate stage

34, 330–334.

of ripening (75% ripe based on peel coloration). This author con- Campos, F.M., Ribeiro, S.M.R., Della Lucia, C.M., Stringheta, P.C., Pinheiro-Sant’Ana,

H.M., 2009. Optimization of methodology to analyze ascorbic acid and dehy-

cluded that fruits used to produce ascorbic acid can be harvested

droascorbic acid in vegetables. Quím. Nova 32, 87–91.

at the green stage after reaching physiological maturity or at an

Castro Neto, M.T., Reinhardt, D.H., 2003. Relations hips between growth parameters

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Cavalini, F.C., Jacomino, A.P., Lochoski, M.A., Kluge, R.A., Ortega, E.M.M., 2006. Matu-

Similar to this study, Bardales et al. (2008) also observed a higher

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ascorbic acid content at the intermediate stage of maturation,

Chirinos, R., Galarza, J., Betalleluz-Pallardel, I., Pedreschi, J., Campos, D., 2010.

which presented a maximum content of 2900 mg/100 g, while the Antioxidant compounds and antioxidant capacity of Peruvian camu-camu (Myr-

green and mature fruits contained approximately 2100 mg/100 g. ciariadubia (H.B.K.) McVaugh) fruit at different maturity stages. Food Chem. 120,

1019–1024.

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Corrêa, M.O.G., Pinto, D.D., Ono, E.O., 2007. Analysis of respiratory activity in jabuti-

in camu-camu fruits during full maturation (1071.12 mg/100 g),

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camu Myrciaria dubia (HBK) Mc Vaugh fruit at four stages of maturation from

camu-camu fruits were green.

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Cunha, A.R., Volpe, C.A., 2011. Growth curves for the fruit of coffee cv. Obatã

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49–62.

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